The present invention relates to a method for the amplification of nucleic acid.
The amplification of specific nucleic acid segments is particularly useful for the generation of detectable amounts of certain kinds of nucleic acid. For example when the presence of nucleic acid characteristic for a specific disease state is to be indicated, this nucleic acid is generally present in biological samples in small amounts. To be able to detect these small amounts of nucleic acid either very sensitive detection methods would have to be used or very large amounts of sample material would have to be concentrated. With the present amplification techniques the small amounts of a specific segment of nucleic acid present in a biological sample can be amplified. This amplified nucleic acid can readily be detected by, for example, hybridizing it to a labelled complementary oligonucleotide. Of course the amplification of nucleic acid is also useful for the generation of larger amounts of nucleic acid used in recombinant DNA techniques and for cloning and sequencing purposes.
Known techniques for the amplification of specific nucleic acid segments are, for example, the polymerase chain reaction (PCR), as described in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202, and Nucleic Acid Sequence Based Amplification ("NASBA"), as described in European Patent application EP 0,329,822.
With PCR large amounts of deoxyribonucleic acid (DNA) are generated by treating a target DNA sequence with oligonucleotide primers such that a primer extension product is synthesized which is separated from the template using heat denaturation and in turn serves as a template, resulting in the amplification of the target DNA sequence. When RNA is to be amplified with PCR the RNA strand is first transcribed into a DNA strand with the aid of reverse transcriptase. Intermediates in the Polymerase Chain Reaction consist of DNA only.
With the aid of "NASBA" large amounts of single stranded RNA are generated from either single stranded RNA or DNA or double stranded DNA. When RNA is to be amplified with "NASBA" the ssRNA serves as a template for the synthesis of a first DNA strand by elongation of a first primer containing a RNA polymerase recognition site. This DNA strand in turn serves as the template for the synthesis of a second, complementary, DNA strand by elongation of a second primer, resulting in a double stranded active RNA-polymerase promoter site, and the second DNA strand serves as a template for the synthesis of large amounts of the first template, the ssRNA, with the aid of a RNA polymerase.
All amplification processes comprise the attachment of primers to templates and subsequent elongation of these primers by certain nucleic acid polymerases that may differ depending on the amplification technique employed.
A problem encountered with amplification of nucleic acid is that nucleic acid is capable of forming various secondary structures. Nucleic acid strands may comprise sequences that may result in the formation of, for example, hairpin loops. These secondary structures might hamper the attachment of a primer to a template and the subsequent elongation of the primer along the template. By interfering with the annealing or extension of the amplification primers these secondary structures lower the efficiency of the amplification.
By the incorporation during amplification of nucleotides that weaken normal base paring the formation of secondary structures, like the formation of internal loops, in the amplificate is prevented. With the incorporation of these structure destabilizing nucleotides secondary structures are destabilized and amplification will become more efficient.
The formation of secondary structures in nucleic acid is also known to be a problem with the sequencing of nucleic acid, because such structures, i.e. compressed regions, may result in anomalous migration patterns during gel electrophoresis. Substitution of inosine for guanosine in the nucleic acid fragments synthesized for the sequencing of RNA has been described by D. R. Mills et al., P.N.A.S., Vol.76, pp.2232-2235, May 1979. With the introduction of inosine in the nucleic acid fragments secondary structures are prevented and the resolution obtained in gel separations after sequencing of the nucleic acid is thereby improved.
The incorporation of a structure destabilizing base analog in DNA amplified with the Polymerase Chain Reaction has been described by Cetus Corporation in PCT application no. WO90/03443. The structure destabilizing nucleotide incorporated during PCR amplification in the method as claimed by Cetus is 7-deaza-2'-deoxyguanosine-5'-triphosphate (c.sup.7 dGTP).
The utilization of c.sup.7 dGTP in PCR results in the incorporation of 7-deazaguanine into the amplified DNA product. This analog differs from normal guanine in that the N-7 of the guanine ring is replaced with a methine moiety which precludes Hoogsteen bond formation. For amplification processes with DNA intermediates the incorporation of c.sup.7 dGTP increases amplification efficiency.